Bio-Rad Cell counter - (Jan/01/2012 )

Dear Guys,
I am working on Tangue squamous cell carcinoma.
I am very new in cell culture techniques (if you have time, please reader my older posts).
I have Bio-Rad cell counting machine, when I am culturing my cells in 6mm plate, and under microscope I saw 70-80% confluency, I try to subculture my cells.
however after trypsinzation, collecting my cells in 3 ml medium, I count them, I have never seen the cell number on my cell counter higher than 50-90 X103 per mililter.
secondly, I was using free sample of bio-rad Trypan blue, however, when I prepared it 0.4% trypan blue solution in 0.81% NaCl and 0.06% KH2PO4, there is a huge difference between my prepared trypan blue, and the one from bio-Rad, as one from bio-rad gives me around 88% viability, and the prepared one give me 8-6% viability for the same sample.

Any advice about use of cell counter and trypan blue.

-madelingirly-

Hi Guys ,

I have added the last photo, I got from my bio-rad cell counter, these blue circle have been putted by my cell counter, .
I wonder the if any one could verify what I could understand from this picture, regarding cells in lumps, which have no blue circles, does this mean that my machine didn`t count it, so I got low cell number despite of confluency because my cells are in a lumps, needs extra pippetting for example.

Best Regards

-madelingirly-

You are correct in thinking that the cells in the clumps have not been counted. You will always get some cells in clumps when subculturing, but from the looks of it, you could probably get a better single cell suspension, though this is sometimes dependent on the cell type, as some cells stick together more than others.

Regarding the lowish cell number - you say 6 mm plate: Did you mean a 96 well plate (each well about 6 mm diameter) or a 6 well plate (each well about 35 mm diameter)? The numbers you are getting are pretty normal for a single well of a 60-80% confluent 6 well plate, but it depends a lot on the cell line. Try looking at the well after you have collected all the cells, and see how much you have left behind - there will always be some cells, so don't worry too much if you see quite a lot.

I would suggest comparing your counts on the cell counter with counts made under a haemocytometer (it is good practice to know how to use these sorts of things).

Regarding your trypan blue - try making it up at 0.05-0.1% in normal PBS and using that. I use 0.4% as a stock solution, rather than as a working solution. It may be that your incorrectly buffered saline solution has an odd pH or something which is causing the cells to die, or that the extra strength of 0.4% is increasing the background, so that the machine is incorrectly reading your samples. You can put some cells on a haemocytometer and look to see if there is actually a difference between the two.

I am very grateful for your helpful advice, I meant 60mm plate, with culturing area around 20 cm2.
I wil try to follow ur advice and use 0.05-0.1% solution and say what will happen.
then take another photo and post it here.
best regards